Diabetes is one of the leading causes of death by disease worldwide. T1D, is a major form of the disease that typically develops at a young age and results from autoimmune destruction of islet beta-cells with consequent insulin deficiency and dependence on exogenous insulin treatment. Apoptosis is the main type of beta-cell death of in T1D. Under normal conditions, maintenance of beta-cell mass is a dynamic process, undergoing both increases and decreases which is dependent on the rates of beta cell survival (i.e. beta-cell proliferation and neogenesis) and beta-cell death (i.e. beta-cell apoptosis and necrosis) thereby control body glucose levels within a narrow physiological range (Bonner-Weir S 2000 Trends Endocrinol Metab, 11:375-378; Bonner-Weir S 2000 Endocrinology 141:1926-1929. In subjects with type 1 diabetes the islet beta-cells are persistently insulted by autoimmune destruction. The beta-cell apoptosis occurs as a result of autoimmune destruction involving T cell infiltration of the islets of Langerhans (Mandrup-Poulsen T 2003 Biochem Pharmacol 66:1433-1440; Mathis D et al, 2001 Nature 414:792-798; Sesti G 2002 Ann Med 34:444-450). The progressive destruction of the pancreatic beta-cells is largely due to lymphocytic infiltration of the islet, resulting in insulin deficiency. Inflammatory cytokines including IL-1beta, TNF-alpha and IFN-gamma are released by macrophages and T cells during this autoimmune response and are important mediators of beta-cell destruction (Mandrup-Poulsen T 2003 Biochem Pharmacol 66:1433-1440; Eizirik D L, Mandrup-Poulsen T 2001 Diabetologia 44:2115-2133; Saldeen J 2000 Endocrinology 141:2003-2010). In the early stage of disease, a compensatory mechanism by which the islet beta-cells attempt expanding to overcome the beta-cell damage caused by autoimmune attack, play a role in maintenance of body's blood glucose. When the rate of beta-cell death significantly exceeds the rate of beta-cell growth, the beta-cells mass is significantly decreased leading to insulin insufficiency and development of diabetic hyperglycemia. Insulin therapy is the major intervention for the treatment of type I diabetes, however, insulin is not a cure as it is hard to manage the exogenous insulin to meet body's needs in a glucose-sensing manner thus to maintain blood glucose levels within a narrow physiological range. Exogenous insulin does not prevent the progression of the disease and severe diabetic complications that eventually arise. Pancreatic islet transplantation is also an effective therapy (Shapiro A M et al, 2000 N Engl J Med 343:230-238) but is limited largely by the limited resources of human islets. In addition, immune-suppressors need to be used for life in the islets-transplanted patients.
At the onset of T1D, over 70-80% of beta-cells have been destroyed (Cnop M et al, 2005 Diabetes 54 Suppl 2:S97-107). The multiple low dose streptozotocin (MLDS) murine model of diabetes is characterized by a progressive hyperglycemia and T-cell mediated beta-cell inflammation (insulitis) similar to that observed in human subjects with recent onset T1D (Rossini A A et al, 1978 Nature. 276(5684):182-4; Harlan D M et al, 1995 Diabetes 44(7):816-23). Another animal model is the non-obese diabetic (NOD) mouse. These mice are an excellent model of autoimmune diabetes (type I diabetes) where islet-antigen reactive T cells infiltrate islets of Langerhans and kill islet beta-cells, and/or initiate an inflammatory process that results in islet beta-cell death (Anderson M S and Bluestone J A 2005 Annu Rev Immunol. 23:447-85). NOD mouse develops spontaneous autoimmune diabetes that shares histoimmunological, serological and clinical features with TID in humans (Tisch R, McDevitt H. 1996 Cell 85(3):291-7; Atkinson M A, Leiter E H. Nat Med. 5(6):601-4). These mice have thus been used extensively and are recognized as the animal model that are mimics type I diabetes in subjects.
Incretins are gastrointestinal hormones that play important role in maintaining body blood glucose levels. The important incretin hormones are glucagon-like peptide-1 (GLP-1) and gastric inhibitory peptide (glucose-dependent insulinotropic peptide or GIP) (Brubaker P L, Drucker D J 2004 Endocrinology 145:2653-2659). Given GLP-1 as an example, this hormonal peptide is secreted from the enteroendocrine L cells of the intestinal tract in response to nutrient ingestion (Hoist J J 1994 Gastroenterology 107:1848-1855; Perfetti R, Merkel P 2000 Eur J Endocrinol 143:717-725). Importantly, GLP-1 is also found to be produced in the islet alpha-cells (Jin T. J 2008 Endocrinol. 198(1):17-28). GLP-1 enhances pancreatic islet beta-cell neogenesis/proliferation and inhibits beta-cell apoptosis; in a glucose-dependent fashion (Nauck M A 2004 Horm Metab Res 36:852-858; Drucker D J 2001 Endocrinology 142:521-527). GLP-1 also augments insulin secretion and lowers blood glucose in rodents as well as in humans in both type I diabetes (17; 18) and type II diabetes Gutniak M et al, 1992 N Engl J Med 326:1316-1322; Dupre J et al J Clin Endocrinol Metab 89:3469-3473). Previous studies have demonstrated that in insulin-secreting beta-cells, the apoptosis and necrosis induced by cytokines could be significantly improved by glucagon-like peptide-1 (GLP-1) or Ex4, a long-acting potent agonist of the GLP-1 receptor (Saldeen J 2000 Endocrinology 141:2003-2010; Li L et al, Diabetologia 48(7):1339-49). In vivo studies have shown that treatment with GLP-1/Ex4, stimulated beta-cell neogenesis in streptozotocin (STZ)-treated newborn rats resulting in persistently improved glucose homeostasis at an adult age (Tourrel C et al, 2001 Diabetes 50:1562-1570). In type I diabetes patients, treatment with Ex4 improved postcibal glycemic excursions (Dupre J et al, 2004 J Clin Endocrinol Metab 89:3469-3473). It is believed that the mechanism by which GLP-1 modulates beta-cell mass involves primarily 1) enhancement of β-cell proliferation, 2) inhibition of apoptosis of β-cells and 3) beta-cell neogenesis (Brubaker P L, Drucker D J 2004 Endocrinology 145:2653-2659).
The biological actions of GLP-1 exert through the activation of the GLP-1 receptor, (GLP-1R). GLP-1R is a member of G-protein coupled receptor (GPCR) superfamily that are expressed mainly by pancreatic beta-cells and to some extent by cells of other tissues (lungs, heart, kidney, GI tract and brain), and is coupled to the cyclic AMP (cAMP) second messenger pathway (Brubaker P L, Drucker D J 2004 Endocrinology 145:2653-2659; Drucker D J 1998 Diabetes 47:159-169). Activation of other protein kinases including Akt (protein kinase B) (Wang Q et al Diabetologia 47:478-487; Brubaker P L, Drucker D J 2004 Endocrinology 145:2653-2659) is found to be important in mediating GLP-1 action in promoting beta-cell growth and inhibiting apoptosis. In animals models of type II diabetes, it has been recently demonstrated that treatment of GLP-1 or Ex4 prevented onset of diabetes (Wang Q, Brubaker P L 2002 Diabetologia 45:1263-1273; Tourrel C, et al, 2002 Diabetes 51:1443-1452) by enhancing beta-cell growth and inhibiting apoptosis (Wang Q, Brubaker P L 2002 Diabetologia 45:1263-1273; Wang Q et al, Diabetologia 47:478-487). GLP-1 has demonstrated clinical efficacy in type II diabetes (Meier J J, Nauck M A 2005 Diabetes Metab Res Rev 21:91-117). It has been demonstrated that expansion of beta-cell mass by treatment with glucagon-like peptide-1 (GLP-1) prevented the onset of diabetes in animal models predisposed to type II diabetes (Wang Q, Brubaker P L 2002 Diabetologia 45:1263-1273; Tourrel C et al, Diabetes 51:1443-1452). U.S. Pat. No. 6,899,883 and U.S. Pat. No. 6,989,148 disclose methods of treating type I diabetes using insulin and glucagon-like peptide 1(7-37) or glucagon-like peptide 1(7-36) amide.
Ex4 is peptide of 39 amino acids that has been isolated from the venom of the lizard Heloderma suspectum (Gila monster) (Eng J et al J Biol Chem. 1992 15; 267(11):7402-5). Ex4, a mammalian homolog does not seem to exist (Pohl M, Wank S A. 1998 J Biol Chem. 17; 273(16):9778-84), shares 53% identity at the amino acid level with the mammalian hormone GLP-1 (Drucker D J 2001 Endocrinology 142:521-527). This peptide displays similar functional features to native GLP-1, including regulation of blood glucose homeostasis, Stimulation of insulin secretion and suppression of glucagon secretion. Ex4 also regulates gastric emptying, food intake. Previous studies showed that Ex4 decreased blood glucose in normal rodents and in animal models of type 2 diabetes (Raufman J P. 1996 Regul Pept. 61(1):1-18). Acting as an agonist of GLP-1 receptor, Ex4, commercially named as Exenatide or Byetta has been approved by the FDA on Apr. 28 2005 for the treatment of type 2 diabetes.
Previous preclinical and clinical studies have showed that GLP-1 or its potent analogue is consistently effective in lowering blood glucose in type 2 diabetes animals and humans. However, GLP-1, is only marginally effective in T1D-prone NOD mice (Hadjiyanni I et al, Endocrinology. 2008 March; 149(3):1338-49). Although Ex4 administered before breakfast also reduced blood glucose in short term studies of human subjects with T1D, likely due to inhibition of glucagon and slowing of gastric emptying (Exendin-4 normalized postcibal glycemic excursions in T1D. (Dupre J, et al 2004 J Clin Endocrinol Metab. 89(7):3469-73). The effects of Ex4 on the prevention of the onset of type 1 animal diabetes appears modest. Study in NOD mice showed that sustained GLP-1R activation was found in the absence of concomitant immune intervention, which was associated only with a transient and modest delay in diabetes onset in these T1D murine models (Hadjiyanni I et al, 2008 Endocrinology 149(3):1338-49), suggesting that GLP-1 alone has very limited effects on the prevention of the development of T1D.
The loss of beta-cell mass in T1D is mainly due to specific autoimmune attack to the islet beta-cells. It is conceivable that a strategy involving promotion of beta-cell growth alone is neither sufficient to prevent the development of T1D nor to effectively treat established T1D in a subject.
Many forms of immunotherapy ameliorate diabetes in NOD mice (Anderson M S, Bluestone J A 2005 Annu Rev Immunol 23:447-485), although most are effective only if initiated prior to the onset of the disease. Unfortunately, most patients initially present with diabetes. More recently, CD3 monoclonal antibody (mAb) therapy was found effective after the onset of disease in NOD mice, and acts by inducing regulatory T cells (Tr) (Belghith M, 2003 Nat Med 9:1202-1208). However, recent clinical trials suggest that CD3 mAb therapy by itself delays beta-cell loss, but cannot return patients to normoglycemia (Herold K C, et al 2005 Diabetes 54:1763-1769). This is presumably because newly diabetic patients have a limited number of residual islet beta-cells, which are not sufficiently protected or replenished by this treatment. Another limitation is that most forms of immunotherapy (as in the case of CD3) are not specific to the autoaggressive T cells, and affect many other immune responses, possibly causing undesirable effects. Notably, administration of ChAglyCD3 (a humanized CD3 mAb) was frequently associated with a cytokine release syndrome and transient Epstein-Barr viral mononucleosis (Keymeulen B et al, 2005 N Engl J Med 352:2598-2608). The failure in the previous clinical trails rationally by tolerizing autoreactive T-cells through administration of islet beta cell antigen (YU L P et al 2002 Ann. N.Y. Acad. Sci. 958: 254-258; Carel J 2002 Engl J Med; 347:1115-1116) suggest that such approaches using tolerizing T-cell alone either did not effectively delay or prevent T1D (Sia C, Homo-Delarche F 2004 Rev Diabet Stud. 1(4):198-206). The findings of these clinical trials using tolerizing autoreactive T-cell strategy also suggest that rather than tolerizing T-cell, the educating T-cell (i.e. suppressing diabetogenic T-cells and enhancing regulatory T-cells) may be of beneficial to maintain the balance of the autoimmune regulation. Therefore, a therapy using the beta-cell growth factor such as GLP-1/Ex4 and combined with an autoimmune suppressor is required to achieve appropriate therapeutic efficacy. In consistent to this notion, recent pre-clinical studies demonstrated that administration of GLP-1/Ex4, combined with immunosuppression by polyclonal anti-T cell antibody (Ogawa N, et al, 2004 Diabetes 53:1700-1705) or anti-CD3 antibody (Exendin-4 improves reversal of diabetes in NOD mice treated with anti-CD3 monoclonal antibody by enhancing recovery of beta-cells. Sherry N A et al 2007 Endocrinology 148(11):5136-44) induced remarkable remission of overtly diabetic NOD mice. However, obvious shortage of this strategy is that the systemic suppression of immunological responses by an anti-T-cell antibody or anti-CD3 antibody may lead to adverse immunologic effects (Keymeulen B, et al, 2005 N Engl J Med 352:2598-2608).
GABA is an important endogenous amino acid synthesized from glutamic acid by glutamate decarboxylase (GAD) (Gottlieb D I. 1988 Sci Am. 258(2):82-9). The role of GABA in the central nervous system has been extensively studied (Gramsbergen J B 2007 J Neurochem. 103(5):1697-708). GABA exerts its biological actions through the activation of its receptors. Three types of GABA receptor, type A, type B and type C (GABAA-CR) are responsible to mediate GABA actions. GABA has long been considered to be the primary inhibitory neurotransmitter in the mammalian brain, and GABAAR is believed to mediate the main GABA inhibitory effects. Whereas under certain circumstance, the excitatory effects of GABA can be mediated by GABAAR (Gramsbergen J B 2007 J Neurochem. December; 103(5):1697-708). During brain development and neuronal maturation GABA acts as a trophic factor (Owens D F, Kriegstein A R. 2002 Nat Rev Neurosci. 3(9):715-27) and modulates neuronal cell proliferation, migration and differentiation (Owens D F, Kriegstein A R. 2002 Nat Rev Neurosci. 3(9):715-27; LoTurco, J J et al, 1995 Neuron 15:1287-1298; Owens, D F et al, 1999 J. Neurophysiol. 82, 570-583).
In the pancreas, GABA is secreted from the islet beta-cells, acting as auto- or paracrine modulator via its receptors in both alpha- and beta-cells (Bansal P, Wang Q. 2008 Am J Physiol Endocrinol Metab. 295(4):E751-61; Franklin I K, Wollheim C B. 2004 J Gen Physiol. March; 123(3):185-90). In particular, GABA suppresses glucagon secretion from alpha-cells through the activation of the GABAA receptor (Rorsman P et al Nature. 1989 341(6239):233-6; Xu E et al, 2006 Cell Metab. 3(1):47-58), while influences beta-cell metabolism and insulin secretion (Murphy K D et al, 2007 J Trauma. 63(5):1099-107; Dong H et al. 2006 Diabetologia 49(4):697-705). In vitro studies demonstrated that GABA promotes beta-cell proliferation inhibits apoptosis (Ligon B et al. 2007 Diabetologia 50(4):764-73). GABA improves metabolism through the modulation of endogenous growth hormones (Murphy K D et al, 2007 J Trauma. 63(5):1099-107). GABAAR are also expressed in T-lymphocytes which provides a fundamental base that GABA can exert direct effects on the T-cells (Alam et al., Mol Immunol, 2006, 43: 1432-42). GABA treatments inhibit the pro-inflammatory T cell response and halt the progression of T1DM in the NOD mouse animal model (Tian et al., 1999 J Neuroimmunol, 96: 21-28; and Tian et al., 2004 J. Immunol., 173:5298-304). In a rat model, GABA has protective effects on streptozotocin-induced diabetes (Nakagawa T, et al, 2005 J Nutr Sci Vitaminol (Tokyo). 51(4):278-82). However, there is no previous art demonstrating that GABA could reverse diabetes after onset of diabetes in a subject.